ABSTRACT Gene expression relies on interplay among cis elements, chromatin domains, and genome architecture. The latter is of intense interest as ~10% of human diseases may arise from defects in genome topology that impact gene expression. Genomes divide into conserved, Mb-sized topologically associated domains (TADs) that are further subdivided into cell type-specific loops between promoter and enhancers (regulatory loops) or between CTCF binding elements (structural loops). In addition, chromatin architecture can be shaped by tissue-specific boundary elements (BEs) that divide active and inactive regions of transcription. These two types of domains tend to associate spatially, perhaps through homotypic chromatin interactions. Foundational questions remain about mechanisms of genome architecture reorganization and its impact on gene expression during cellular differentiation. Answers to these questions have important implications because disease-associated variants in the human genome can disrupt CTCF sites or BEs, enabling aberrant communication between enhancers and alternative promoters that normally partition into separate architectural domains. The co-PIs have approached relationships between genome topology and gene regulation by focusing on the mouse Tcrb antigen receptor locus for several reasons, including: (i) it is a physiological model of manageable complexity (ii) its architecture and transcription are dynamically regulated during T cell development, (iii) it divides into alternating chromatin domains, (iv) changes in topology and transcription are critical for Tcrb assembly by long-range recombination, and (v) its recombination center (RC) has a simple regulatory landscape with one enhancer that communicates with two promoters to initiate all aspects of Tcrb assembly. The PIs' recent collaborations have provided important clues into the dynamics of Tcrb structure at a low level of resolution, but insights into mechanisms that sculpt the observed architectural changes are still lacking. These and other data support their hypothesis that developmental switches between inactive and active Tcrb conformations are orchestrated by tissue- and stage-specific changes in the binding of CTCF to cornerstone elements and by the transcription status of individual gene segments, which cooperate to compartmentalize Tcrb into distinct structural domains and drive homotypic interactions that facilitate long-range Tcrb gene assembly. To test foundational aspects of their hypothesis, the PIs propose to elucidate detailed topologies of active versus inactive Tcrb loci (Aim 1), assess whether transcription status and homotypic chromatin interactions shape Tcrb conformations (Aim 2), and determine mechanisms by which CTCF elements direct Tcrb topology (Aim 3). The co-PIs will monitor multiple physiological readouts (topology, transcription, chromatin, and recombination) to gain unprecedented insights into mechanistic relationships among genome architecture, gene expression, DNA recombination, and factors that sculpt primary lymphocyte antigen receptor gene repertoires